The objectives of this project are to identify and solve the technical and neurobiological problems associated with the growth in vitro of two dimensional neuronal networks of over 30 neurons in which all interconnections are known, all neurons can be stimulated and all major extracellular activity can be monitored for an extended period of time. We have established reliable UV laser microbeam cell surgery techniques to carry out "network tailoring" in closed chambers. The technique was developed with neuroblastoma cells in year 1 of this grant period, confirmed on neurons in year two and will be explored further (pathological limits to UV surgery) in year three. We have developed multimicroelectrode surfaces with 36 photoetched microelectrodes integrated into the floor of a tissue culture chamber and will be utilizing new plates containing 50 microelectrodes. We have recorded with these photoetched electrodes from mouse spinal neurons and plan a systematic analysis of simultaneous, long term activity in small CNS tissue fragments, cell aggregates and single neurons. We will also simplify the cell aggregates with laser surgery until a defined network is created. This network will be analyzed with emphasis on network morphological stability, longevity, spontaneous activity and culture requirments.